Project 3: Project Summary/Abstract The overall goal of Project 3 is to modulate the levels of anti-HIV-1 chimeric antigen receptor (CAR) and broadly neutralizing antibodies (bNAb) modified immune cells by developing the most effective and safe positive and negative selection strategy to (1) achieve a therapeutic level of repopulation and (2) incorporate a safety ?kill- switch? to eliminate the genetically engineered anti-HIV-1 immune effector cells in cases of unexpected adverse effects, such as cytokine storm, autoimmune reaction and malignant transformation. The hematopoietic stem cell based gene therapy approach has shown great promise to achieve an HIV-1 cure. However, one of the major limitations has been the difficulty of achieving the engraftment levels sufficient to provide therapeutic efficacy, in particular for HIV-1 infected patients where intensive myeloablative conditionings would be an unfavorable risk-benefit. Thus, a safe and titratable positive selection strategy is highly desirable to maximize the level of anti-HIV-1 gene engineered immune cells to treat patients with HIV-1 without dangerous intensive myeloablation. Furthermore, it is important to incorporate a safety ?kill-switch? procedure to eliminate the genetically engineered anti-HIV-1 immune effector cells based on lessons learned from severe adverse effects in cancer immunotherapy. Therefore, we will develop a negative selection strategy as a safety ?kill-switch? to eliminate genetically engineered immune cells. We will identify the most effective and safe selection strategy from (1) knocking down hypoxanthine-guanine phosphoribosyltransferase (HPRT) expression using RNA interference that enables us to effectively enrich or eliminate anti-HIV-1 gene-modified HSPC using clinically available prodrug 6-thioguanine or methotrexate, (2) co-expressing truncated non-functional human epidermal growth factor receptor (huEGFRt), a cell surface marker for a rapid ex vivo positive selection and in vivo negative selection by a FDA-approved anti-EGFR monoclonal antibody Cetuximab (Erbitux) and (3) the P140K mutant form of human O6-methylguanine-DNA- methyltransferase (MGMTP140K) for a positive selection. We hypothesize that a clinically relevant, safe and effective positive and negative selection strategy can be developed by rigorously evaluating our proposed selection strategies for our anti-HIV-1 CAR and scFv-Fc bNAb combining therapies to achieve a cure of HIV-1 disease.